Freely Available Online INTERNATIONAL JOURNAL OF LIMNOLOGY ISSN NO: 2691-3208

Research DOI: 10.14302/issn.2691-3208.ijli-18-2476

The Possibility of Using the Fresh Water Bivalve, Spathopsis Rubens, in The Nile River, El Mahmoudia Water Stream As Bioindicator For Pollution

E H Radwan1,*, A Abdel Mawgood2, A Z Ghonim1, R El Nagar1

1Faculty of Science, Damanhour University, Egypt 2Institute of Graduate Studies and Environmental Research, Damanhour University, Egypt

Abstract Bivalves are used as bioindicators of heavy metals pollution because they are known to concentrate these elements, providing a time integrated indication of environmental contamination. Trace metals can reach high concentrations in sediments and also in aquatic organisms by bioaccumulation through the food chain. Six heavy metals (Hg, Zn, Pb, Fe, Mg and Cu) were collected and investigated from Abu Hummus, El Behara. The concentration of Hg was high in winter as 2.3µg/g in sediment. The Zn concentration was high in summer in sediment as 8.1µg/g. The Pb concentration was high in winter in water as 3.3µg/l. The concentration of Fe in sediment was high in summer as 492 µg/g. The concentration of Mg was high in sediment as 408µg/g. The concentration of Cu was high in summer in sediment as 301µg/g. The mean concentrations of Fe in the present study are within the permissible limits of law 48/1982 (<1 mg/l) and the guideline of (WHO, 1993) which is <1 mg/l. The mean concentration level of copper is within the permissible limits of law 48/1982 (<1.0 mg/l). The mean levels of the heavy metals (Hg, Zn, Pb, Fe, Mg and Cu) detected in the present study in the water stream are less than the permissible limits recommended by (USEPA, 2005). In the present study there is a significance between all seasons in the protein content in the soft tissue of Spathopsis rubens as the mean concentration level in Spring was reported as 102.83mg/g which is higher then that of autumn 100.5mg/g, summer 93.33 mg/ g and winter 80.50 mg/g. In the present study the mean activity level of GPx in spring was higher than the other seasons such as spring 31.33u/g ˃ summer 28.33 u/g ˃Autumn 26.67 u/g ˃ winter 20.50u/g. The mean activity level of SOD in summer was higher than the other seasons such as summer 38.83 u/g ˃ spring 33.33 U /g ˃Autumn 28.83U/g ˃ winter 22.83U/g. The mean activity level of CAT in spring was higher than the other seasons such as spring 25.67u/g ˃ summer and autumn19.83u/g ˃ winter 15.17u/g. The mean activity level of MDA in winter was 30.50 U/g ˃ summer 22.50U/g ˃ autumn 18.0 U/g ˃ spring 16.83U/g. In the present study it was found that the mean activity level of MDA increased in winter at the same time the mean activity level of CAT, SOD and GPx were decreased in winter. Negative correlation was reported between CAT and Hg in winter as r=-0.88*. A positive correlation coefficient in winter was found between SOD activity level and CAT activity level as r=0.838*.

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Corresponding Author: E H Radwan, Faculty of Science, Damanhour University, Egypt, Email: [email protected] Keywords: Bivalve-heavy metals-fresh water-Abo Homous-ElMahmoudia stream. Received: Nov 10, 2018 Accepted: Dec 16, 2018 Published: Dec 22, 2018 Editor: Patricio De los Ríos, Universidad Católica de Temuco, Chile.

Introduction population dynamics of the gastropods which play an important role in the health of man and his livestock [8]. The Nile River is a source of life to millions of Ali [9] illustrated that molluscs are suitable candidates to people. Pollution caused by inadequate drainage be used in biomonitoring surveys of Lake Qarun in systems in rural villages, and irrigation wastewater filled Egypt. Freshwater bivalves provide many ecological with fertilizers and pesticides. Different analytical services to aquatic systems [10, 11]. Large invertebrates methods were constructed to monitor the water quality can be considered metabolic reactors because they status in freshwater ecosystems [1]. The Nile River transfer nutrients and energy from water to sediments water is facing environmental and public health by filtering and nutrient mineralization [10, 12]. The problems of water pollution which affects water quality study on mollusk as a possible bioindicator of river water and influences the balance of the whole ecosystem [2]. quality is because of the fact that they have the ability The rapid progress in industry led to the release of to concentrate pollutants as they are filter feeders [13]. heavy metals in the ecosystem and especially the fresh Industrial effluents contributing to aquatic pollution water ecosystem. The accumulation of heavy metals in contain toxic substances which include heavy metals. the Nile River water affects the quality of the water. The Indiscriminate discharges of these wastes alter the iron and steel industry releases lead and zinc into the quality of water and cause hazards to the fauna. Copper Nile River. Amer and Abdel Gawad [3] monitored the is a micro-nutrient and is present as a metal ion in distribution of heavy metals in the Nile River water and certain enzymes and plays an important role in the studied the impacts of heavy metals on the water transfer of electrons in electron transport chain. It is a quality. Bakhiet [4] and Ayodele and Abubakar [5] component of haemocyanin. There is an increased body suggested that the study of heavy metal contamination of evidence implicating heavy metals as a potential in bivalves is important in order to consider them as threat to aquatic organism by way of studies on their bioindicators for heavy metal contamination.The physiology, biochemistry and ecology. Marine organisms pollutants are carried from the source and tend to sink are characterized by a greater spatial ability to thereby polluting the aquatic environment. Although accumulate some metals [14]. Marine organisms are information on contaminated regions in the tropical characterized by a greater spatial ability to accumulate areas are lacking, studies on pollution monitoring in some metals when compared with bottom fresh water lakes environment have been reported using sediments [15]. The shellfish represents an important different indicator species [6, 5]. Freshwater mollusc source of protein for coastal communities. Over 90% of communities are important in terms of biodiversity and human health exposure to several contaminants occurs ecosystem health. They play significant roles in the through diet primarily seafood [16, 17]. In order to public and veterinary health and thus need to be evaluate the adverse effect of the pollutants on aquatic scientifically more extensively [7]. organisms, there is a world wide trend to complement A lot of researchers studied the ecology and physical and chemical parameter with biomarkers in

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aquatic pollution monitoring [18, 19]. of the highly reactive superoxide anion to O2 and to the Since bivalves are filter feeders, they concentrate less reactive species H2O2. Peroxide can be destroyed by contaminants to a much higher level than those of the CAT or GPx reactions [32]. Among the biomarker of surrounding sea [20]. These contaminants may cause stress, the primary key events in oxidative damage are diseases of humans, especially microbial contaminants, lipid peroxidation (MDA) [33-36]. because shellfish are often eaten raw or lightly Aim of the work: Spathopsis rubens had been cooked [21, 22]. To reduce the risk, the source of the choosen as example of bivalve which lives in El Behara shellfish should be investigated and better quality would governerate fresh water, to study the levels of heavy be attained by appropriate treatment following the metals such as Zn, Cu, Pb, Mg, Fe and Hg in water, harvest. The effects of environmental contaminants may sediment and flesh of Spathopsis rubens collected from result from direct toxic actions on tissues or cells or from El Mahmodia stream, River Nile. The aim of the present alterations of the homeostatic mechanisms including the study is to establish its suitability as bio-indicator that immune system [23-28]. The protein content in the could be used to monitor heavy metals pollution in Nile tissues of plays a role in the metabolism of River and to determine CAT, SOD, GPx and the potential animals [29]. Heavy metals mainly react with proteins of lipid peroxidation. To know the effect of pollutants on and adversely alter the physiological activities hence Biochemicals (protein, lipid and Carbohydrates) in cause risk of life in different way. Protein acts as Spathopsis rubens. enzyme, hormone and basic structural component of the Material and Methods . Protein is key substance to show the effect of In December 2016 to July 2017 the selected heavy metal. Proteins respond to stress condition for bivales were collected from Abu Hummus, River Nile, El better survival by altering their levels. The shellfish Beheira Egypt (Figure.1). The shell sizes of the detected represents an important source of protein for coastal samples were ranging from (10–15 cm) in length, from communities. It has been predictable, for instance, that (6–9 cm) in width and from (2.6 to 4.5 cm) in height. over 90% of human health exposure to several The sediment and water samples were collected in contaminants occurs through diet primarily corresponding to the clam settlements to determine the seafood [15-17]. initial level of heavy metals. Contamination of fresh water with a wide range Samples of Spathopsis rubens were collected of pollutants has become a matter of concern over last from Abu Hummus El Beheira, Egypt. Abu hummus lies few decades. The defence mechanisms against free between the Cairo-Alexandria Agricultural road and the radical-induced oxidative damage include the following El Mahmodea stream at; 31.10063oN-30.310063oE. The catalytic removal of free radicals and reactive species by water samples, sediment and flesh of Spathopsis rubens factors such as CAT, SOD, GPx. Animal CAT areheme- were collected from the river water side. Water samples containing enzymes that convert hydrogen peroxide were collected in plastic bottles, pre-rinsed with distilled (H2O2) to water and O2, and they are largely localized in water. The bivalves were chosen by harvesting only subcellular organelles such as peroxisomes. large but with similar sizes and healthy. A total Mitochondria and the endoplasmic reticulum contain of [15-20] samples were collected/location/season then little CAT. The intracellular H2O2 cannot be eliminated were kept in plastic containers filled with water. unless it diffuses to the peroxisomes [30]. GSH-Px The biochemical analysis includes the removes H2O2 by coupling its reduction with the oxidation of GSH. GSH-Px can also reduce other determination of metal analysis, organic pollutants, peroxides. Most animal tissues contain both CAT and protein, lipids, carbohydrates and antioxidant enzymes GSH-Px activity. SODs are metal-containing proteins that (CAT, SOD, GPx and MDA). The analysis of heavy metals catalyze the removal of superoxide, generating water (Cu, Fe, Mg, Zn, Pb and Hg) of fresh water was done peroxide as a final product of the dismutation [31]. SOD according to Ayodele and Abubakar [5]. The heavy is the antioxidant enzyme that catalysed the dismutation metals in sediment and in soft tissues were measured www.openaccesspub.org IJLI CC-license DOI: 10.14302/issn.2691-3208.ijli-18-2476 Vol-1 Issue -1 Pg. no.– 3

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Figure 1. The map of Abu Hummus, ElBehara Figure 2. Spathopsis rubens (Google map).

according to [37]. The results are presented as mean ± been choosen (Figure. 2) in the present study. S.D. values. One-way analysis of variance (ANOVA) was Results used to test the significance of depuration in each metal Bivalve samples were collected from their concentration and TPHs. Post hoc test was used to natural beds from Abu Hummus, El Behirea, Egypt.The analyse the multiple comparisons among water, survey in the present study was reported as the sediment and soft parts. All statistical analyses were following: Spathopsis wahlbergi hartmanni (Martens, performed using the SPSS 15.0 software [38]. 1866), Spathopsis rubens arcuata (Cailliaud, 1823), Determination of carbohydrate and lipids were according Caelatura (Horusia) parreyssi (Philippi, 1847), Lanistes to [39]. Determination of protein was estimated by carinates (Olivier, 1804) and Melanoides tuberculata Lowry’s method [40]. Determination of Catalase activity (Müller, 1774), Melanoides tuberculata (Müller, 1774), (CAT) was measured according to Aebigh [41]. Lanistes carinates (Olivier, 1804), Mutela singularis Superoxide dismutase (SOD, EC 1.15.1.1) activity was (Pallary, 1924), Caelatura (Caelatura) prasidens measured using the procedure of Beauchamp and (Cailliaud, 1827). The Spathopsis rubens had been Fridovich [42, 43]. Glutathione peroxidase activity levels choosen (Figure. 2) in the present study. (Tables 1 - 4) were determined by the method of Pagtia and Valentine [44]. Lipid peroxidation (Malondialdehyde) was The mean concentration level of Hg was higher determined by the method of OhKawa [45]. in winter in sediment as 2.3µg/g than in water and in tissue. Results The mean concentration level of Zn was higher Bivalve samples were collected from their in summer in sediment as 8.1 than in water and tissue. natural beds from Abu Hummus, El Behirea, Egypt.The The mean concentration level of Pb was higher in winter survey in the present study was reported as the in water as 3.3µg/g than in sediment and tissue. The following: Spathopsis wahlbergi hartmanni (Martens, mean concentration level of Fe was higher in summer as 1866), Spathopsis rubens arcuata (Cailliaud, 1823), 492µg/g than in winter and tissue. The Mg Lanistes carinates (Olivier, 1804) and Melanoides concentrations were higher in sediment as 408µg/g than tuberculata (Müller, 1774), Melanoides tuberculata in water and tissue. The Cu concentrations were higher (Müller, 1774), Lanistes carinates (Olivier, 1804), Mutela in summer in sediment as 301µg/g than in water and singularis (Pallary, 1924). The Spathopsis rubens had tissue. www.openaccesspub.org IJLI CC-license DOI: 10.14302/issn.2691-3208.ijli-18-2476 Vol-1 Issue -1 Pg. no.– 4

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Table 1. The mean concentration levels of heavy metals in water, sediment and bivalve tissue collected in autumn (2016-2017)

Sediment µg/g Tissue (µg/g) Heavy metals Water (no 1), µg/L (no 1) Mean (no 6)

Hg 1.1 2.1 0.93

Zn 3.1 4.2 2.08

Pb 2.7 2.2 1.73

Fe 300 417 299.7

Mg 2.44 3.7 2.61

Cu 1.95 2.2 1.35

Table 2. The mean concentration levels of heavy metals in water, sediment and bivalve tissue collected in Winter (2016-2017)

Heavy metals Water µg/L Sediment µg/g Mean of tissue µg/g

Hg 1.8 2.3 1.46

Zn 3.9 4.5 2.17

Pb 3.3 2.5 1.65

Fe 292 392 322

Mg 2.6 3.5 1.73

Cu 2.1 2 1.66

Table 3. The mean concentration levels of heavy metals in water, sediment and bivalve tissue collected in Spring (2016-2017)

Water µg/L Sediment µg/g Average (no 6) Heavy metals (no 1) (no 1) tissue µg/g

Hg 1.5 1.9 1.26

Zn 4.1 5.2 2.83

Pb 2.2 2.6 1.32

Fe 235 400 252.33

Mg 3.2 4.1 2.38

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Table 4. The mean concentration levels of heavy metals in water, sediment and bivalve tissue collected in Summer (2016-2017)

Heavy metals Water µg/L(no 1) Sediment µg/g (No 1) tissue µg/L(no 6)

Hg 1.6 2.2 1.10 Zn 5.6 8.1 4.23 Pb 2.8 2.9 0.89 Fe 321 492 274.2 Mg 4.1 4.8 2.21 Cu 2.1 3.1 1.14

Table 5. The mean activity level of GPx Table (6): The mean activity level of SOD during during (2016-2017). (2016-2017).

GPx Mean ± SD. SOD Mean ± SD. Autumn (n = 6) 26.67 ± 6.35 Autumn (n= 6) 28.83bc ± 5.67

Winter (n = 6) 20.50 ± 4.85 Winter (n = 6) 22.83c± 4.36 Spring (n = 6) 31.33 ± 6.35 Spring (n = 6) 33.33ab± 7.81 Summer (n = 6) 28.33 ± 9.09 Summer (n = 6) 38.83a± 8.64 F (p) 2.681 (0.074) F (p) 5.919* (0.005*) Means with different letters are significant; F, p: F and p values for ANOVA test. Means with different letters are significant; F, p: Signeficance between groups was done F and p values for ANOVA test. Signeficance using Post Hoc Test (LSD). *: Statistically between groups was done using Post Hoc Test significant at p ≤ 0.05 (LSD). *: Statistically significant at p ≤ 0.05

Table 7. The mean activity level of CAT during (2016-2017).

CAT Mean ± SD. Autumn (n = 6) 19.83 ± 5.46 Winter (n = 6) 15.17 ± 5.38 Spring (n = 6) 25.67 ± 8.80 Summer (n = 6) 19.83 ± 3.76 F (p) 2.951 (0.057)

Means with different letters are significant; F, p: F and p values for ANOVA test. Signeficance between groups was done using Post Hoc Test (LSD). *: Statistically significant at p ≤ 0.05 www.openaccesspub.org IJLI CC-license DOI: 10.14302/issn.2691-3208.ijli-18-2476 Vol-1 Issue -1 Pg. no.– 6

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Table 8. The mean activity level of MDA Table 9. The total protein content in during (2016-2017). Protein Mean ± SD. MDA Mean ± SD. Autumn (n = 6) 100.5 ± 15.04 Autumn (n = 6) 18.0b ± 2.83

a Winter (n = 6) 80.50 ± 12.10 Winter (n = 6) 30.50 ± 8.26

Spring (n = 6) 16.83b± 6.94 Spring (n = 6) 102.83 ± 18.67

Summer (n = 6) 22.50b± 6.28 Summer (n = 6) 93.33 ± 13.7 F (p) 5.620* (0.006*) F (p) 2.657 (0.076)

Means with different letters are significant; F, p: F and p values for ANOVA test. Signeficance between Means with different letters are significant.F,p: F groups was done using Post Hoc Test (LSD). *: and p values for ANOVA test, Significant between Statistically significant at p ≤ 0.05 groups was done using Post Hoc Test (LSD).*: Statistically significant at p ≤ 0.05

Table 10. The total lipid content in Table 11. The carbohydrates content in

Lipid Mean ± SD. Carbohydrates Mean ± SD.

Autumn (n = 6) 12.78 ± 2.34 Autumn (n = 6) 12.05 ± 1.91

Winter (n = 6) 10.60 ± 2.90 Winter (n = 6) 10.62 ± 2.50

Spring (n = 6) 10.25 ± 1.20 Spring (n = 6) 13.40 ± 2.72

Summer (n = 6) 11.78 ± 3.63 Summer (n = 6) 9.38 ± 1.54

F (p) 2.837 (0.064) F(p) 1.029(0.401)

Means with different letters are significant.F,p: F Means with different letters are significant.F,p: F and p values for ANOVA test, Significant between and p values for ANOVA test, Significant between groups was done using Post Hoc Test (LSD).*: groups was done using Post Hoc Test (LSD).*: Statistically significant at p ≤ 0.05 Statistically significant at p ≤ 0.05

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Figure 3. The mean concentration of the activity levels of Figure 4. The mean concentration of the activity GPx (mU/mg.protein) in different seasons levels of SOD (U/g.tissue) in different seasons (2016-2017). (2016-2017).

Figure 5. The mean concentration levels of Figure 6. The mean concentration levels of activity of CAT(U/g) in different seasons during activity of MDA (nmol/mg tissue) in different (2016-2017). seasons (2016-2017).

Figure 7. The mean concentration levels of total Protein Figure 8. The mean concentration levels of lipid (g/dl) in different seasons during (2016-2017). (mg/dl) in different seasons during (2016-2017).

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Figure 9. The mean concentration levels of carbohydrates in different seasons during (2016-2017).

A Histogram of the mean activity level of concentration level of Fe and Mg in tissues showed a different enzymes in the bivalve (2016-2017) negative correlation as r=-0.835*. (Figures 3 - 6) In spring only the mean concentration level of A histogram representing the selected Pb and Cu in tissue showed a high significant correlation biochemical parameters in the bivalve (2016-2017) as r=0.978*. (Figures 7 - 9) (Tables 5 - 11) In summer there were positive correlation A histogram of the mean concentration levels of between the mean activity level of GPx and lipid content the selected heavy metals in water (µg/g) in different and Hg in tissues as r=0.837* and seasons during the year (2016-2017) (Figures 10 - 15) r=0.865*; respectively. The mean activity level of MDA (Tables 12 - 16) was positively correlated with the mean concentration In Autumn the activity of GPx and of MDA were level of Fe in tissues as r=0.821*. The mean level of the positively correlated with the carbohydrate contents in total protein content was positively correlated with Pb the bivalve as r=0.956* an r=0.865*; respectively. The mean concentration level as r=0.893* and negatively activity level of SOD is negatively correlated with MDA, correlated with Cu mean concentration level Hg, Fe as r=-0.873*, r=-0.998*, r=-0.925*; as r=-0.912*. Whereas the mean concentration level of respectively. The activity of CAT is negatively correlated the lipid content was negatively correlated with the with the lipid content, Pb as r=-0.922*, r=-0.87*; mean concentration level of the carbohydrate contents respectively. as r=-0.828* and Pb in tissue was also negatively correlated with Cu in tissues as r=-0.985. The total protein content is negatively correlated with Fe concentration level in tissues Discussion as r=-0.908*. The lipid contents is positively correlated In the present study Spathopsis rubens was with the carbohydrate contents as r=0.877* and collected from El Beheira, Egypt, Abu Hummus. These r=0.910*; respectively. Both Hg and Pb are positively species was already detected in previous reports [46]. In correlated with Fe concentration level in tissues of the the present study the heavy metals (Hg, Zn, Pb, Fe, Cu bivalve as r=0.932* and r=0.856*; respectively. and Mg) were detected in the four seasons from autumn The correlation coefficient in winter was only (2016) to summer (2017). In fresh water, in sediment between SOD mean activity inhibition level and CAT and in the soft tissues of Spathopsis rubens. Some mean activity level as r=0.838*. Negative correlation enzyme activities were detected as; CAT, SOD, GPx and was found between CAT and Hg as r=0.88*. The mean MDA. The total protein, Lipid and carbohydrates in the soft tissues of Spathposis rubens were detected. There

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Figure 10. the mean concentration level of Hg Figure 11. the mean concentration level of Zn in the different seasons during the year in the different seasons during the year (2016-2017). (2016-2017).

Figure 12. the mean concentration level of Pb Figure 13. the mean concentration level of Fe in different seasons during the year in different seasons during the year (2016-2017). (2016-2017).

Figure 14. the mean concentration level of Figure 15. the mean concentration level of Cu Mg in different seasons during the year in different seasons during the year (2016-2017). (2016-2017).

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Table 12. The mean concentration levels of heavy metals in the tissue (Zn, Pb, Fe, Mg, Cu and Hg) in different seasons during the year ( 2016-2017):

Zn Mean ± S.D. Pb Mean ± S.D.

a Autumn (n = 6) 2.08b ± 0.52 Autumn (n = 6) 1.73 ± 0.37

a Winter (n = 6) 2.17b± 0.63 Winter (n = 6) 1.65 ± 0.34

a Spring (n = 6) 2.83b± 0.82 Spring (n = 6) 1.32 ± 0.40

Summer (n = 6) 4.23a± 0.67 Summer (n = 6) 0.89b± 0.29

F (p) 13.300* (<0.001*) F (p) 7.032* (0.002*)

Fe Mean ± S.D. Mg Mean ± S.D.

Autumn (n = 6) 299.67 ± 52.82 Autumn (n = 6) 2.61a ± 0.65

b Winter (n = 6) 322.33 ± 67.30 Winter (n = 6) 1.73 ± 0.45

a Spring (n = 6) 252.33 ± 29.97 Spring (n = 6) 2.38 ± 0.30

Summer (n = 6) 274.17 ± 45.59 Summer (n = 6) 2.21ab± 0.32

F (p) 2.157 (0.12) F(p) 4.043* (0.021*)

Cu Mean ± SD. Hg Mean ± SD.

Autumn (n = 6) 1.35 ± 0.31 Autumn (n = 6) 0.93b ± 0.21

Winter (n = 6) 1.66 ± 0.29 Winter (n = 6) 1.46a± 0.45

Spring (n = 6) 1.41 ± 0.32 Spring (n = 6) 1.26ab± 0.21

Summer (n = 6) 1.14 ± 0.34 Summer (n = 6) 1.10b± 0.19

F (p) 2.7 (0.07) F (p) 3.756* (0.027*)

F, p: F and p values for ANOVA test, Significant between groups was done using Post Hoc Test (LSD). *: Statistically significant at p ≤ 0.05.

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Table 13. Correlation between different studied parameters in Autumn (2016-2017)

Carbohy­ SOD CAT MDA Protein Lipid Hg Zn Pb Fe Mg Cu drates

r 0.070 -0.655 -0.033 0.189 0.714 0.956* -0.117 -0.009 0.444 0.050 0.691 0.764 GPx p 0.895 0.158 0.950 0.721 0.111 0.003 0.825 0.987 0.378 0.925 0.128 0.077

r 0.303 -0.873* 0.744 -0.544 -0.167 -0.998* 0.200 -0.639 -0.925* -0.534 0.003 SOD p 0.560 0.023 0.090 0.265 0.751 <0.001 0.704 0.172 0.008 0.275 0.996

r -0.052 0.481 -0.922* -0.805 -0.296 -0.579 -0.870* -0.536 -0.432 -0.581 CAT

p 0.922 0.334 0.009 0.054 0.570 0.228 0.024 0.273 0.393 0.227

r -0.588 0.374 0.148 0.865* -0.503 0.483 0.796 0.521 0.157 MDA p 0.220 0.465 0.779 0.026 0.309 0.332 0.058 0.289 0.766

r -0.499 -0.106 -0.775 -0.364 -0.793 -0.908* -0.020 -0.189 Protein p 0.314 0.841 0.070 0.479 0.060 0.012 0.970 0.719

r 0.877* 0.522 0.234 0.910* 0.692 0.714 0.619 Lipid p 0.022 0.289 0.655 0.012 0.128 0.111 0.190

Carbo­ r 0.128 0.084 0.685 0.329 0.724 0.821 hydrate s p 0.809 0.874 0.133 0.524 0.104 0.045

r -0.161 0.639 0.932* 0.482 -0.028 Hg p 0.761 0.172 0.007 0.333 0.958

r 0.365 0.072 -0.453 0.069 Zn p 0.477 0.892 0.367 0.897

r 0.856* 0.449 0.606 Pb p 0.029 0.371 0.202

r 0.426 0.262 Fe p 0.400 0.616

r 0.365 Mg p 0.477

r Cu p

r: Pearson coefficient *: Statistically significant at p ≤ 0.05

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Table 14. Correlation between different studied parameters in Winter (2016-2017)

Carbohy­ SOD CAT MDA Protein Lipid Hg Zn Pb Fe Mg Cu drates r 0.800 0.632 -0.542 0.421 0.169 0.217 -0.714 -0.079 0.055 -0.564 0.284 -0.227 GPx p 0.056 0.178 0.267 0.405 0.748 0.680 0.111 0.882 0.918 0.244 0.585 0.665 r 0.838* -0.570 0.290 -0.035 -0.404 -0.664 0.202 -0.142 -0.108 0.075 -0.605 SOD p 0.037 0.238 0.577 0.948 0.427 0.150 0.701 0.788 0.839 0.888 0.203 r -0.456 0.032 0.190 -0.314 -0.880* -0.146 0.236 0.159 -0.160 -0.318 CAT p 0.363 0.952 0.719 0.545 0.021 0.783 0.653 0.763 0.763 0.538 r -0.339 -0.606 0.136 0.604 -0.408 0.517 0.211 -0.226 0.479 MDA p 0.511 0.202 0.797 0.205 0.422 0.293 0.688 0.667 0.337 r -0.286 0.116 -0.027 0.608 -0.017 -0.035 -0.393 0.185 Protein p 0.583 0.827 0.960 0.200 0.974 0.947 0.441 0.725 r 0.345 -0.570 -0.309 -0.145 -0.311 0.408 -0.029 Lipid p 0.503 0.238 0.552 0.785 0.548 0.422 0.956 Carbo­ r -0.090 -0.550 0.440 -0.614 0.238 0.705 hydrate p 0.865 0.258 0.383 0.195 0.650 0.118 s r 0.329 -0.258 0.134 -0.046 0.122 Hg p 0.525 0.622 0.801 0.931 0.818 r -0.657 0.277 -0.243 -0.406 Zn p 0.156 0.595 0.643 0.425 r 0.212 -0.511 0.768 Pb p 0.687 0.300 0.074 r -0.835* 0.105 Fe p 0.039 0.844 r -0.474 Mg p 0.343 r Cu p

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Table 15. Correlation between different studied parameters in Spring (2016-2017):

Carbohy­ SOD CAT MDA Protein Lipid Hg Zn Pb Fe Mg Cu drates - r 0.235 0.407 0.138 -0.754 -0.274 -0.765 0.069 0.649 0.293 -0.324 0.529 GPx 0.590 P 0.654 0.423 0.795 0.083 0.600 0.076 0.897 0.218 0.163 0.573 0.530 0.281 - R 0.357 0.709 -0.274 0.248 0.011 0.090 0.023 -0.288 0.165 -0.457 SOD 0.079 P 0.488 0.115 0.600 0.636 0.983 0.866 0.965 0.580 0.882 0.754 0.363 - R 0.778 -0.779 0.594 0.030 0.774 0.316 0.392 0.258 0.224 CAT 0.513 P 0.068 0.068 0.214 0.955 0.071 0.298 0.542 0.442 0.621 0.670 - R -0.621 0.790 0.242 0.391 0.096 0.119 0.236 -0.028 MDA 0.317 P 0.188 0.061 0.644 0.443 0.541 0.857 0.823 0.653 0.958 - R -0.406 0.431 -0.265 0.700 -0.773 0.240 -0.652 Protein 0.434 P 0.425 0.393 0.612 0.122 0.071 0.390 0.647 0.160 - R 0.372 0.291 0.108 0.320 0.048 0.060 Lipid 0.056 P 0.468 0.576 0.917 0.838 0.536 0.929 0.911 - Carbohy­ R 0.316 0.050 -0.523 0.804 -0.413 0.584 drates P 0.541 0.926 0.287 0.223 0.054 0.415 - R -0.129 0.208 0.616 -0.130 Hg 0.243 P 0.643 0.808 0.692 0.192 0.805 R -0.727 0.225 -0.226 -0.731 Zn P 0.101 0.668 0.667 0.099 R 0.296 -0.471 0.978* Pb P 0.569 0.346 0.001 R -0.622 0.201 Fe P 0.187 0.702 R -0.395 Mg P 0.438 R Cu P

r: Pearson coefficient *: Statistically significant at p ≤ 0.05

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Table 16. Correlation between different studied parameters in Summer (2016-2017)

Carbo­ SOD CAT MDA Protein Lipid hydrate Hg Zn Pb Fe Mg Cu s r -0.106 -0.395 -0.185 -0.095 0.837* -0.667 0.865* -0.113 -0.022 -0.338 0.390 0.038 GPx P 0.841 0.438 0.725 0.858 0.038 0.148 0.026 0.831 0.968 0.512 0.445 0.942 r -0.309 0.437 0.693 -0.198 0.544 0.108 0.637 0.363 0.574 0.204 -0.371 SOD P 0.552 0.387 0.127 0.706 0.264 0.839 0.174 0.479 0.234 0.699 0.469 r 0.579 -0.419 -0.662 0.274 -0.350 0.192 -0.583 0.584 -0.386 0.528 CAT P 0.228 0.408 0.152 0.600 0.497 0.716 0.224 0.223 0.450 0.281 r 0.030 -0.451 0.251 0.061 0.363 -0.310 0.821* 0.216 0.331 MDA P 0.955 0.370 0.631 0.909 0.479 0.549 0.045 0.681 0.521 r -0.239 0.482 -0.267 0.268 0.893* 0.176 0.367 -0.912* Protein P 0.649 0.333 0.609 0.608 0.016 0.739 0.474 0.011 r -0.823* 0.806 -0.403 -0.021 -0.673 0.393 0.093 Lipid P 0.044 0.053 0.428 0.969 0.143 0.441 0.861 r -0.592 0.752 0.181 0.697 -0.520 -0.294 Carbohydrates P 0.216 0.085 0.732 0.124 0.290 0.572 r 0.065 -0.316 -0.112 0.296 0.353 Hg P 0.902 0.542 0.833 0.569 0.492 r -0.145 0.797 -0.508 0.017 Zn P 0.785 0.057 0.304 0.974 r -0.265 0.473 -0.985* Pb P 0.612 0.344 <0.001

r -0.255 0.191 Iron P 0.625 0.717 r -0.339 Mg P 0.511 r Cu P

r: Pearson coefficient. Statistically significant at p ≤ 0.05

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is a general acceptance that fresh water ecosystems 1.32µg/g˃ summer 0.89µg/g, Mg in autumn 2.6 µg/g˃ undergo little ecological stress when subjected to spring 2.38µg/g ˃ summer 2.4µg/g˃ Autumn 1.73µg/g. salinities up to 1000 mgL-1. Much of the knowledge of The mean concentration level of Zn is higher in summer the impacts of salinity on aquatic ecosystems comes than the other seasons as, Zn in summer 4.23 µg/g˃ from field sampling a long a gradient of salinity from spring 2.83µg/g ˃ winter 2.17µg/g˃ autumn 2.08µg/g. which it is difficult attribute cause of ecological The present observation showed that the mean change [47]. concentrations level of heavy metal in the sediment Bivalves have been used as bioindicators of were high when compared with standard values [48]. pollution because they have the ability to concentrate The mean concentration level of Hg in sediment is heavy metals to several other magnitudes [48]. The higher in winter than the other seasons as, winter 2.3 mean concentrations of Fe in the present study are µg/g ˃ summer 2.2 µg/g ˃Autumn 2.1 µg/g ˃ spring within the permissible limits of law 48/1982 (<1 mg/l) 1.9 µg/g.The mean concentration level of Zn, Cu, Pb, Fe and the guideline of [49] which is <1 mg/l. The mean and Mg were higher in summer than the other seasons concentration level of copper is within the permissible as; Zn in summer 8.1 µg/g ˃ spring 5.2 µg/g winter 4.5 limits of law 48/1982 (<1.0 mg/l), the values of the µg/g ˃ Autumn 4.2 µg/g, Cu in summer 3.1 µg/g ˃ measured metal. The mean levels of the heavy metals in spring 2.3 µg/g ˃ Autumn 2.2 µg/g ˃ winter 2 µg/g, Pb water are less than that of the permissible limits in summer 2.9 µg/g ˃ spring 2.6 µg/g ˃ winter 2.5 µg/ recommended by [50]. The changes in metabolic rates g˃Autumn 2.2 µg/g. Fe in summer 492 µg/g ˃ Autumn of bivalves within the seasons and the variation in 417 µg/gram ˃ spring 400 µg/gram ˃ winter 395 µg/g, bioavailability of metals in the surrounding environment Mg in summer 4.8 µg/gram ˃ spring 4.1 µg/gram ˃ with time might be responsible for the health status of Autumn 3.7 µg/gram ˃ winter 3.5 µg/g.The variations in the molluscs [51]. The present study is in agreement metal concentration of the shellfish tissues in the with Cossa and Rondeau [51] in that the higher metal present study could be related to the concentration of burden and concentration in the wet season (such as for heavy metals in the fresh water. Fe and Zn). Lower levels for Cd and Hg could be Abdulah [53] reported that the high level of attributed to wash out of the lagoons during the rainy heavy metals in the lake may be related to their period. Biological variables such as changes in the tissue concentration in the stream and rivers discharging into composition as well as the season of sampling and the the lake. The high level of Zn, Cu and Pb in the river hydrodynamics of the lagoons have to be considered. indicates the quality of the water prevailing at the period Seasonal variations are related to a great extent to of sampling. Trace metal concentrations in clams depend seasonal changes in flesh weight during development of on numerous environmental and biological factors [54]. gonadic tissues [52]. Earlier studies by Chouba et al. [55] in Tunisia The present observation showed that the mean demonstrated higher concentrations of heavy metals in concentration levels of Cu, Hg and Fe in tissues are clams during high rainfall periods. These findings are in higher in winter than the other seasons as; the mean agreement with that of the present study. Studies have concentration level of Hg in winter 1.46 µg/g ˃ spring shown that during the spawning period, proteins and 1.26µg/g ˃ summer 1.1 µg/g˃ autumn 0.93µg/g. The carbohydrate contents, which have a high affinity for mean concentration level of Cu in winter 1.6 µg/g˃ heavy metals, are accumulated for gonad tissue spring 1.4µg/g ˃ summer 1.14µg/g˃ Autumn 1.35µg/g production, energetic storage and consumption and the mean concentration level of Fe in winter 322.33 [56].There is no access waste water treatment in Abo µg/g˃ Autumn 299.67µg/g ˃ summer 274.17 µg/g˃ Hummus rural areas as 20% of Egyptian villages have spring 252.33µg/g .The mean concentration levels of Pb inadequate potable water [57-58]. and Mg are higher in autumn than the other seasons as Pollution of the aquatic environment by Pb in Autumn 1.73 µg/g˃ winter 1.65µg/g ˃ spring inorganic and organic chemicals is a major factor posing

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a serious threat to the survival of aquatic metals and this may be the reason for inactivation of the organisms [59]. The aquatic environment is subjected to enzyme, involved in the carbohydrate metabolism [73]. various types of pollutants which enter water In the present study the higher concentration of bodies [60]. It is estimated that the total amount of carbohydrates was found in spring 13.40 mg/gm than in reused treated wastewater in Egypt was about 1.4 billion autumn 12.05 mg/gm, summer 11.78 mg/gm and in m3 in 2000 [61]. Industrial waste water is considered winter 10.62mg/gm. the second of the main sources of Nile water pollution. Free radicals are able to react with biological Effluent wastewater is often partially treated [62]. Major macromolecules and produce enzyme activation, lipid pollutants in agricultural drains are salts, nutrients, peroxidation [74]. Antioxidant enzymes activity levels of pesticide residues, pathogens and toxic organic and marine bivalve Perna viridis during heavy metals inorganic pollutants [63, 64]. At high pollution stress exposure were significantly higher in tissues. The mantle however, protein synthesis can be suppressed indicating was observed to significantly contribute to the disturbance of normal metabolic processes [65-67]. The organismal response to lipid peroxidation as indicated by fall in protein level during pollutant exposure may be high activity levels of antioxidant enzymes [75]. Cu due to increased catabolism and decrease in protein strongly stimulates the lipid peroxidation damage of the synthesis [68]. The digestive gland is the main site of gill plasma membranes [76, 77]. Pannunzio and degradation and detoxification of toxicants and hence Storey [78] observed a suppression of GPx activity resulting into increasing utilization of protein to meet during anoxia exposure in the hepatopancreas of the energy demand. The higher degradation of protein is the marine gastropods Littorina littorea. Main enzymes tool to access the extent of toxicity [69]. involved in detoxification from reactive oxygen species. In the present study there is a significance SOD and GPx have been shown to contribute to between all seasons in the protein content in the soft antioxidant defense in the mussels [79]. Glutathione is tissue of Spathopsis rubens as the mean concentration considered a scavenger able to protect cells from level in Spring was reported as 102.83mg/g which is oxidative damage [80, 81]. Aerobic organisms are higher then that of autumn 100.5 mg/g, summer 93.33 protected against oxidative stress by antioxidant mg/g and winter 80.50 mg/g. Kharat et al. [70] studied systems which mobilis enzymes such as the (Cu-Zn

depletion in protein content in the tissues of superoxide dismutase) which transfers O2 to H2O2 [82]. Macrobrachium kistnensis exposed to different Oxidative stress induced by copper exposure, concentrations of tributyltin chloride stress on protein evidenced by increased lipid peroxidation products such metabolism similar results were obtained by Sole and as malondi aldehyde has also been demonstrated for the Porte [71]. Inhibition in the protein synthesis was mussels Mytilus galloprovincialis [83], Perna perna [84], reported to be due to non-selective blocking of Ruditapes decussatus [85], and for the oyster phosphorylation process in the central nervous virginica [86]. Antioxidant defenses may be system [72]. Bivalves generally store carbohydrates in increased or inhibited by chemical stressors. The large amounts during their growing seasons and use occurrence of one kind of response or the other depends them over the rest of the year although proteins may be on the intensity and duration of the applied stress and an energy reserve in some bivalve species. Lipids have the susceptibility of the species that are exposed [87]. been reported to function most importantly as energy There are several reports on increased SOD and CAT storage substances and physical properties of biological activities in bivalves in the presence of excess free membranes. In the present study the higher radicals [88]. Dietary copper appears to be innocuous to concentration of lipid in autumn 12.78 mg/g than in the digestive system at low concentrations as copper is winter 10.60 mg/g, spring 10.25 mg/g and in summer a cofactor of enzymes such as cytosolic 9.38mg/g .Fall in carbohydrates level may be due to the SOD (Cu-SODiso-enzyme) [89] and is also part of the prolonged exposure of the metabolism to the heavy hemocyanin molecule. The excess of this metal could be

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sequestered into vacuoles or immobilized by biological 2. Anwar WA (2003). Environmental health in Egypt. compounds for a possible excretion [90, 91]. Metals can Int J Hygiene Environ Health 206:339-350. induce oxyradical production leading to lipid 3. Amer AS, Abdel Gawad HA (2012). Rapid peroxidation [92]. In the present study the mean bioindicators assessment of macrobiotic pollution on activity level of GPx in Spring was higher than the other aquatic environment. Int Water Technol J seasons such as spring 31.33U/g ˃ summer 28.33 2(3):196 –206. U/g ˃autumn 26.67 U/g ˃ winter 20.50U/g. The mean 4. Bekhiet (2015). Determination of Heavy Metals in activity level of SOD in summer was higher than the Fish Tissues and Water from White Nile Khartoum other seasons such as summer 38.83 U/g ˃ spring 33.33 City – Sudan J of Environ Protect and Sustainable U/g ˃autumn 28.83U/g ˃ winter 22.83U/g. The mean Development Vol. 1, No. 3, 2015, pp. 178-181. activity level of CAT in spring was higher than the other seasons such as spring 25.67U/g ˃ summer and 5. Ayodele JT and Abubakar MB (2001). Cleopatra autumn19.83U/g ˃ winter 15.17U/g. The mean activity bulimode and Mutelarubens as bio-indicators of level of MDA in winter was 30.50 U/g ˃ summer heavy metals in river wudil, Kano Nigeria Research 22.50U/g ˃ autumn 18.0 U/g ˃spring 16.83U/g. In the Journal of Science, 7: Nos. 1 and 2. present study it was found that the mean activity level 6. Ayodele JT and Abu Bakar MB (2000). Trace metal of MDA increased in winter at the same time the mean levels in river wudil, Kano Nigeria Trop. J of Environ. activity level of CAT, SOD and GPx were decreased in Res. 3:230-237. winter. 7. Supian Z and Ikhwanuddin AM (2002). Population Conclusion dynamics of freshwater molluscs (Gastropod: Fe, Hg and Cu are higher in winter season while Melanoides tuberculata) in Crocker Range Park, Pb, Zn and Mg are higher in summer in tissue of Sabah. ASEAN Review of Biodiversity and Spathopsis rubens. Pb and Hg are higher in winter Environmental Conservation (ARBEC). season, Zn, Fe and Mg are higher in summerWhile Cu is 8. Mostafa (2009). Effect of salinity and drought on the higher in summer and winter in fresh water. Fe, Zn, Cu, survival of Biomphalaria arabica, the intermediate Pb and Mg are higher in summer while Hg is higher in host of Schistosoma mansoni in Saudi Arabia winter in sediment. Egyptian Academic Journal of Biological Science, 1 The high ratio of protein and carbohydrates in (1) (2009), pp. 1-6. spring while the higher ratio of lipids in autumn.CAT, 9. Ali M, Abdel-Meguid M, Abdin A (2006). Assessment GPx are higher in spring, SOD is higher in summer while of floating fish cages impacts on the water, fauna, MDA is higher in winter. By the effect of aquaculture flora, sediments, aquatic weeds, fish and hydraulics activities, irrigation, mechanized farming and future of Damieta branch, Scientific Bulletin, Faculty of increased loading of agro-industrial effluents and Engineering, Helwan University April 2006. domestic waste, The pollution increase in winter due to 10. Spooner DE, Vaughn CC (2006). Context-dependent rain water (winter) which move pollutants to river Nile effects of freshwater mussels on stream benthic and the effect of pollutants appear in Spathopsis rubens communities. Freshwater Biology 51, 1016–1021. on the following season, this effect has a disturbance of 11. Zimmerman GF, de Szalay FA (2007). Influence of ecosystem and food chains in the aquatic environment. unionid mussels (: Unionidae) on sediment References stability: an artificial stream study. Fundamental and 1. El-Sheekh MM (2016). River Nile pollutants and their Applied Limnology 168, 299–306. effect on life forms and water quality. In: Dumont 12. Van Ginneken L, Chowdhury MJ, Blust R (1999). HJ (ed) The Nile: origin, environments, limnology Bioavailability of cadmium and zinc to the common and human use. Springer International publishing carp, Cyprinuscarpio, in complexing environments: a Hdb Env chem, DOi10.1007/698-2016-97. test for the validity of the free ion activity model. www.openaccesspub.org IJLI CC-license DOI: 10.14302/issn.2691-3208.ijli-18-2476 Vol-1 Issue -1 Pg. no.– 18

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